F. M. Cunningham

Early neutrophil but not eosinophil or platelet recruitment to the lungs of allergic horses following antigen exposure

Previous studies have shown that bronchoalveolar lavage fluid from horses with allergic respiratory disease and showing clinical symptoms contains increased numbers of neutrophils. In some cases, the eosinophil count is also increased. In this study the time course of changes in lung function and the accumulation of radiolabelled leucocytes and platelets in the lungs of allergic and normal horses has been examined during a 7 hr allergen exposure. Antigen challenge had no effect on pleural pressure or the distribution of radiolabelled neutrophils, eosinophils or platelets in normal horses. In contrast, in 6/8 allergic horses, there was an increase in pleural pressure and neutrophil accumulation in the lungs, both of which were evident after 4–5 hr. However, during the 7 hr challenge period radiolabelled eosinophils were detected in the lungs of only 1/6 horses exhibiting an increase in pleural pressure and in 1/7 horses that failed to show a change in airway function despite a clinical history of allergic respiratory disease. Antigen challenge did not alter the distribution of radiolabelled platelets in the five allergic horses tested. These results demonstrate that increased pleural pressure is not accompanied by eosinophil or platelet accumulation in the lungs of horses with allergic respiratory disease following exposure to antigen. However, changes in airway function can be associated with neutrophil accumulation but can also take place in the absence of this cell recruitment. This raises the possibility that the presence of neutrophils in the lung is not a prerequisite for changes in lung function.

Retrospective study of the relationships between age, inflammation and the isolation of bacteria from the lower respiratory tract of thoroughbred horses.

A total of 1235 tracheal aspirates taken from 724 thoroughbreds in race training, aged from two to 10 years, were examined cytologically and bacteriologically. An inflammation scoring system on a scale of 0 to 9 was devised to allow the severity of lower airway disease to be assessed from the cytological results. The inflammation scores were closely related to the isolation of bacteria (P<0.001), and the most common bacterial isolates were Streptococcus zooepidemicus, Streptococcus pneumoniae and Pasteurella/Actinobacillus-like species. Lower airway disease was less common in older horses (P=0.031), and the groups at highest risk were the two- and four-year-olds. Lower airway inflammation was more common in the four-year-olds at National Hunt yards than in the four-year-olds at flat racing yards (P=0.040, odds ratio=3.80).

Phenotypic and functional characterization of a CD4(+) CD25(high) FOXP3(high) regulatory T-cell population in the dog

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross‐reactive anti‐mouse/rat Foxp3 antibody clone FJK‐16s to identify a population of canine CD4+ FOXP3high T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3+ cells in both PB and LN yielded positive staining with the newly developed anti‐murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4+ and CD8+ T cells. Removal of the Con A and continued culture disclosed a CD4+ FOXP3high population, distinct from the CD4+ FOXP3intermediate T cells; very few CD8+ FOXP3high T cells were observed, though CD8+ FOXP3intermediate cells were present in equal abundance to CD4+ FOXP3intermediate cells. The CD4+ FOXP3high T cells were thought to represent activated Treg cells, in contrast to the FOXP3intermediate cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co‐staining with interferon‐γ (IFN‐γ) supported this notion, because the FOXP3high T cells were almost exclusively IFN‐γ−, whereas the FOXP3intermediate cells expressed a more heterogeneous IFN‐γ phenotype. Following activation of mononuclear cells with Con A and interleukin‐2, the 5% of CD4+ T cells showing the highest CD25 expression (CD4+ CD25high) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4+ T cells with the lowest CD25 expression (CD4+ CD25−), which proliferated readily. The CD4+ CD25high FOXP3high T cells were able to suppress the proliferation of responder CD4+ T cells in vitro, in contrast to the CD4+ CD25− cells, which showed no regulatory properties.

Report of the 3rd Havemeyer Workshop on allergic diseases of the horse, Hólar, Iceland, June 2007

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

Endotoxin and dietary amines may increase plasma 5‐hydroxytryptamine in the horse

Uptake of 5-hydroxytryptamine (5-HT) into platelets is an important mechanism by which low plasma concentrations are maintained, and platelet activation may therefore result in significant release of this vasoconstrictor. The present study examined the kinetics of active uptake of radiolabelled [3H]5-HT by washed equine platelets in vitro, and investigated the effects on this process of 4 other naturally occurring monoamines which may be released from the caecum in conditions of carbohydrate overload. The release of [3H]5-HT by platelets was also studied, since platelet accumulation and activation has been associated with acute laminitis. Release of [3H]5-HT was measured in response to platelet activating factor (PAF), unlabelled 5-HT and the indirect activation of platelets by endotoxin in the presence of blood leucocytes. Km value for the uptake of 5-HT by equine platelets was 2.4 +/- 0.6 micromol/l and the Vmax was 8.3 +/- 0.6 pmol [3H]5-HT/10(7) platelets/min. The rate of uptake of 5 micromol/l [3H]5-HT was significantly decreased by the uptake inhibitors fluvoxamine and clomipramine. The 4 other monoamines examined all inhibited the uptake of [3H]5-HT in a noncompetitive manner, decreasing Vmax by between 17 and 82%. Incubation of platelets with LPS (0.1 mg/ml) in the absence of leucocytes did not result in significant release of [3H]5-HT; however, in the presence of leucocytes 3.8 +/- 1.7 pmol [3H]5-HT/10(7) platelets (mean +/- s.e.) were released. This release was significantly inhibited by parthenolide and WEB2086, but not by aspirin. This suggests that PAF from activated leucocytes was responsible for the 5-HT release. These data show that 5-HT uptake by equine platelets is a saturable process operating most efficiently at substrate concentrations in the low micromolar range. The noncompetitive inhibition of 5-HT uptake by other naturally occurring monoamines may result in increased plasma concentrations of 5-HT, as would its release by endotoxin. Such a rise in plasma 5-HT concentrations may contribute to selective vasoconstriction in the equine digital circulation.

Differential superoxide anion generation by equine eosinophils and neutrophils.

Equine eosinophils and neutrophils are believed to play an important part in the protection of horses against parasitic and bacterial invasion. Eosinophils may also play a key role in the pathogenesis of equine inflammatory conditions such as the allergic skin disease, insect hypersensitivity. The factors which stimulate the respiratory burst of equine eosinophils and neutrophils are poorly understood. The first aim of the present study was to determine the effects of the phorbol ester, phorbol myristate acetate (PMA), which is believed to activate intracellular protein kinase C, and opsonised particles of serum-treated zymosan (STZ), on the production of superoxide anions by equine eosinophils and neutrophils. Since histamine has been detected after antigen challenge in the skin of horses with insect hypersensitivity, the second aim was to establish the effects of this mediator on superoxide anion production by equine eosinophils and the receptor sub-type(s) that mediate histamine-induced responses. For comparison, responses of neutrophils from the same horses were also examined. PMA and STZ induced significant increases in superoxide anion generation by equine eosinophils and neutrophils. The estimated maximum (EMAX) superoxide anion production by eosinophils in the presence of PMA was significantly greater than that of neutrophils; the estimated concentration of PMA inducing 50% of the maximum response (EC50) by eosinophils was significantly less. The EMAX values for superoxide anion production by neutrophils in the presence of STZ were significantly greater than those for eosinophils. Histamine induced superoxide anion generation by equine eosinophils which was inhibited by the histamine-1 receptor antagonists chlorpheniramine and mepyramine, but not the histamine-2 and histamine-3 receptor antagonists, cimetidine and thioperamide, respectively. Histamine did not cause superoxide anion production by equine neutrophils. These studies demonstrate that equine granulocytes vary in their ability to produce a respiratory burst in the presence of different stimuli, with eosinophils being more responsive to protein kinase C activators and neutrophils to opsonised particles. They also show that histamine selectively induced the generation of superoxide anions by equine eosinophils via histamine-1 receptor activation. Thus, in horses with insect hypersensitivity, histamine released from cutaneous mast cells after antigen challenge could activate eosinophils which have migrated into the dermis.

Platelet activating factor mimics antigen-induced cutaneous inflammatory responses in sweet itch horses

Hypersensitivity responses to biting flies such as Culicoides are believed to be the cause of sweet itch, a seasonal intensely pruritic skin condition of horses. Little is known about the mediators released by antigen in the skin of affected horses. In the present study the cutaneous vascular and cellular responses to intradermally injected platelet activating factor (PAF) have been characterised in sweet itch cases during the active phase of the disease and compared with those of Culicoides antigen extract. Histamine was used as a positive control in vascular permeability studies. Responses were also examined in 4 of the 5 sweet itch cases during the inactive phase of the disease. Normal ponies were used as controls. PAF-induced increases in vascular permeability that were dose-related (0.001-1 micrograms per site) and of a similar magnitude in sweet itch and normal animals. Antigen (0.5-50 micrograms per site) also caused dose-related wheal formation in sweet itch cases during the active, but not the inactive, phase of the disease. This effect was biphasic, with maximal responses occurring at 1 and 8 h. An increase in vascular permeability occurred in normal ponies only after administration of the highest dose of antigen tested. Interestingly, histamine (0.02 micrograms per site) induced wheals were significantly smaller in the affected, compared with the normal, group, both during the active and inactive phases. PAF and antigen caused neutrophil accumulation in the skin of sweet itch and normal animals during both the active and inactive phases of the disease. Eosinophil recruitment was also observed but only in the affected group and, in the case of PAF, during the active, but not the inactive, phase. Antigen additionally caused the accumulation of mononuclear cells in the skin of sweet itch cases during the active phase, PAF induced a small increase in mononuclear cell numbers in these animals but the increase was not statistically significant. These findings demonstrate that PAF mimics the effects of Culicoides antigen during the active phase of the disease. Hence, PAF, like histamine, may play a role in the pathogenesis of antigen-induced responses in the skin of sweet itch horses.

Cloning of equine chemokines eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, mRNA expression in tissues and induction by IL-4 in dermal fibroblasts.

We report the cloning of four equine CC chemokines, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, which show high levels of identity with their respective homologous sequences in other species. Using a multiplex RT-PCR, we have studied the constitutive mRNA expression of these four CC chemokines in skin, lung, liver, spleen, jejunum, colon and kidney of normal adult horses and compared this data with the eosinophil counts in the same samples. We demonstrate that eotaxin mRNA is only expressed in jejunum and colon, where there are large numbers of eosinophils suggesting that eotaxin might be recruiting eosinophils in the normal digestive tract of the horse. MCP-1 and MCP-4 are expressed in all tissues whereas MCP-2 is only found in some samples of lung, spleen, liver and kidney. We also report the early induction (2h) of equine eotaxin and MCP-4, and the up-regulation of MCP-1 by interleukin-4 in dermal fibroblasts, suggesting these chemokines might be involved in equine skin allergic diseases.

Inhaled leukotrienes cause bronchoconstriction and neutrophil accumulation in horses.

Leukotrienes have been shown to mimic many of the pathophysiological processes in allergic airway disease. In this study the bronchoconstrictor effect of inhaled LTD4, and radiolabelled neutrophil accumulation in response to inhalation of LTB4, have been examined in the horse. In separate studies, solutions of LTD4 and LTB4 were administered to the airways of normal animals by nebulisation. LTD4, but not LTB4, caused a dose-dependent increase in pleural pressure which was maximal at three to four minutes and had returned to baseline by 15 to 20 minutes. On a molar basis LTD4 was 305 to 970 times more potent than methacholine. LTB4 induced an early recruitment (15 minutes to 1 hour) to the lungs of radiolabelled neutrophils, which persisted for more than 5 hours in some animals. There was no effect on peripheral blood leucocyte counts or pleural pressure and neither LTB4, nor LTD4, affected respiratory rate. These results suggest that, if released during antigen challenge, LTB4 and LTD4 could contribute to the pathogenesis of equine COPD. In a small group of asymptomatic COPD horses these leukotrienes appeared to cause similar, but smaller, changes in lung function and neutrophil recruitment, which could suggest reduced responsiveness to these mediators.

Inhibition of antigen-induced cutaneous responses of ponies with insect hypersensitivity by the histamine-1 receptor antagonist chlorpheniramine

A whole-body extract of Culicoides impunctatus induced a biphasic increase in oedema formation in ponies with insect hypersensitivity, with maxima after one and eight hours. The Culicoides antigen did not induce similar responses in ponies with no previous history of the disease. In insect-hypersensitive ponies the local administration of chlorpheniramine (12 μg) completely inhibited oedema formation in response to histamine (0.04 μg) and to Culicoides antigen (0.5 μg) at one hour, and the response to Culicoides antigen at eight hours was inhibited by 63 per cent. Chlorpheniramine also partially inhibited the accumulation of eosinophils and neutrophils induced by Culicoides antigen after two hours.